Chemistry: molecular biology and microbiology – Treatment of micro-organisms or enzymes with electrical or... – Cell membrane or cell surface is target
Reexamination Certificate
1999-05-27
2002-11-05
Weber, Jon P. (Department: 1651)
Chemistry: molecular biology and microbiology
Treatment of micro-organisms or enzymes with electrical or...
Cell membrane or cell surface is target
C435S173400
Reexamination Certificate
active
06475760
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention concerns a method for intracellular manipulation of at least one deposited biological cell situated in a cultural medium adhering to a support area, wherein an opening is created in the membrane of the cell, and the cell interior is manipulated through this opening. The invention furthermore relates to an apparatus for manipulation of the cell interior of at least one biological cell having a cell membrane situated in a culture medium. The apparatus has an object carrier, which has at least one support area on which the cell is adherently depositable, a poration tool for opening the cell membrane, and at least one entry channel situated in the area of the poration tool for manipulation of the cell interior.
An apparatus of the type mentioned at the beginning is already known from Alberts, B. et al.,
Molecular Biology of the Cell
, Third Printing, VCH Verlag (1995), page 212 ff, which has a hollow needle connected with a suction device and made of an electrical insulating material having an interior cavity which has an opening on the free end of the hollow needle. For opening the cell membrane, the opening situated at the free end of the hollow needle is set upon the exterior of the cell membrane in order then to create an underpressure in the interior cavity of the hollow needle by means of the suction device. Through this underpressure, a cell membrane piece situated in front of the opening of the hollow needle is torn out of the membrane formation. After introducing the opening into the cell membrane the hollow needle, engaging the edge of the cell membrane surrounding the opening, electrically insulates the cell fluid contained in the interior of the cell against the culture medium. The interior of the cell is manipulated through the opening introduced into the cell membrane. For example, the cell nucleus can be removed by sucking cell fluid into the hollow needle from the cell, and subsequently another cell nucleus can be installed through the opening into the cell interior.
The previously known process and the apparatus for implementing the process have the disadvantage that a micromanipulator is necessary for positioning the hollow needle on the cell. This results in a comparatively more complicated and expensive device. Moreover, the accessibility of the cells situated on the object carrier by the micromanipulator is greatly reduced. The process and the apparatus are, for this reason, only suited for an intracellular manipulation of individual, or at best for a simultaneous manipulation of a small number of cells situated on the object carrier. At the same time, a costly, manual positioning of the hollow needle on the cell is necessary.
Arranging a cell floating in a culture medium between two large surface area electrodes, respectively spaced at a distance from the cell, and applying an electric voltage to these electrodes is also already known from Alberts, B. et al.,
Molecular Biology of the Cell
, Third printing, VCH Verlag (1995), page 213. Moreover, the membrane of the cell is simultaneously opened at several places by electroporation so that the genetic material situated in the culture medium to be introduced into the cell can be diffused into the cell interior through the openings of the cell. In this connection, however, it is disadvantageous that such an introduction of genetic material is subject to statistical fluctuations and is influenced by various parameters such as the concentration of genetic material in the area of the cell, the size of the genetic material to be introduced, and the size of the openings created in the cell membrane. The previously known process consequently does allow any selective manipulation of the cell interior.
SUMMARY OF THE INVENTION
There thus exists an object of creating a process and an apparatus of the type mentioned at the beginning, which make a simple manipulation of the interior of a cell possible. In particular, an expensive manual positioning of a hollow needle on the cell to be processed is to be avoided. This objective is accomplished according to the process of the invention in that the opening is introduced into the cell membrane within the support area of the cell and at a distance from its support edge.
In this manner, it is possible to arrange a poration agent for creating the cell membrane opening or a poration tool in the support area of the cell on the object carrier, so that when the cell is deposited on the support area, it is at the same time positioned on the poration agent or the poration tool. In this manner, an expensive manual positioning of a poration tool can be omitted. Since the opening is introduced into the cell membrane within the support area of the cell and at a distance from the edge of the support area, the membrane area of the cell surrounding the opening and adhering to the support area for the cell seals the opening against the culture medium. The cell fluid situated in the interior of the cell is thereby electrically insulated against the culture medium as extensively as possible, so that a potential equilization between the cell potential in the interior of the cell and that of the culture medium is prevented. Through the opening created into the cell membrane, the cell interior can be manipulated.
With an especially advantageous embodiment of the invention, the opening is introduced into the cell membrane by means of electroporation. While conducting the process, the electroporation electrode can, for example, be arranged in the support area on which the adhering cell lies . To introduce the opening into the cell membrane, only an electrical voltage then needs to be applied between the electroporation electrode and the culture medium. This brings about a flow of electrical current which opens the cell membrane.
With another embodiment of the invention, at least one mechanical impulse is exerted to introduce the opening into the cell membrane on a partial area of the cell membrane. In this connection, this partial area is released from the membrane formation. Optionally, an impulse succession can also be applied with several individual impulses.
It is especially advantageous if the opening is introduced into the cell membrane by means of sound waves, especially ultrasound and/or hypersound waves. Here, it is even possible for the sound waves to be focused on the area of the cell membrane to be opened and/or for several sound waves to be superimposed, such that their oscillations in the area of the cell membrane are overlaid into an oscillation with increased amplitude. The cell membrane can thereby be opened without contact.
A contact-free opening of the cell membrane can, however, also take place in that a partial area of the cell membrane is irradiated with energy-rich radiation, especially with laser radiation. In this connection, the wave length of the radiation is preferably selected such that the cell membrane absorbs the radiation well. Expediently, the radiation is launched into the cell at its support area. Optionally, however, a laser beam can also be launched into the cell outside the support area, in that first of all a small launch opening is introduced in a membrane area situated there. The laser beam is subsequently projected through the opening and through the interior of the cell to a membrane area situated in the support area of the cell, in order to cut out a partial area of the membrane from the membrane formation by swivelling the laser beam around the launch opening.
With another embodiment of the process, the opening is introduced into the cell membrane by the action of a chemical poration substance. Performing or Triton®, for example, can be used as poration agents.
It is especially advantageous if an electrical and/or chemical and/or a radiation-activatable chemical substance is used, and if this substance for introducing the opening into the cell membrane is activated by the action of radiation, a chemical and/or an electrical field. The substance is thus activated by supplying energy. In this manner, free radicals
Baumann Werner
Brischwein Martin
Ehret Ralf
Freund Ingo
Gahle Hans-Jürgen
Akin Gump Strauss Hauer & Feld L.L.P.
Micronas GmbH
Weber Jon P.
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